Central Role In Autophagy Research Articles

Theaflavin-3,3'-Digallate Ameliorates Collagen-Induced Arthritis Through Regulation of Autophagy and Macrophage Polarization.

Previous studies have presented that theaflavin-3,3'-digallate (TFDG), one of natural flavonoids, have protective effects on collagen-induced arthritis (CIA). Besides, it was reported that TFDG could affect inflammatory signaling pathways, like NF-κB, JNK, and so on, to ameliorate inflammation. However, the anti-inflammatory mechanisms mentioned above are common to natural flavonoid products including TFDG. Therefore, this study aimed to further investigate the other mechanisms of TFDG against CIA. DBA/1 mice (8-10 weeks) were intravenously injected Freund's Adjuvant (100μL) at the base of tail and intraperitoneally injected PBS or different dosage of TFDG (1 mg/kg or 10 mg/kg). Then the paw and knee tissues were collected to assess the severity of joint destruction. In vitro experiments, bone marrow macrophages (BMMs) were exposed to TNF-α (10ng/mL) with or without different concentrations of TFDG (0.1μmol/L or 1.0μmol/L). Besides, the targets of TFDG were predicted with docking software and were verified through experiment. TFDG treatment could reduce M1 macrophage (pro-inflammatory) and inflammatory cytokines, such as IL-1, IL- 6 and TNF-α, both in vitro and in vivo. At the same time, the M2 macrophage (alternatively activated) polarization was promoted by TFDG. Animal experiments showed TFDG ameliorated joint destructions. For investigating the mechanisms, the targets of TFDG were predicted by bioinformatics tools. According to predictions, we hypothesized that TFDG could act with BCL-2 to weaken the interaction between BCL-2 and Beclin1. Beclin1 plays a central role in autophagy, and we found that the autophagy level of BMMs was recovered by TFDG. Besides, 3-MA, an autophagy inhibitor, could attenuate the therapeutic effect of TFDG. TFDG protected against collagen-induced arthritis by attenuating the inflammation and promoting anti-inflammatory M2 macrophage polarization through controlling autophagy.

Exploring autophagy-related prognostic genes of Alzheimer's disease based on pathway crosstalk analysis.

Recent studies have shown that different signaling pathways are involved in the pathogenesis of Alzheimer’s disease (AD), with complex molecular connections existing between these pathways. Autophagy is crucial for the degradation and production of pathogenic proteins in AD, and it shows link with other AD-related pathways. However, the current methods for identifying potential therapeutic targets for AD are primarily based on single-gene analysis or a single signal pathway, both of which are somewhat limited. Finding other methods are necessary for providing novel underlying AD therapeutic targets. Therefore, given the central role of autophagy in AD and its interplay with its pathways, we aimed to identify prognostic genes related to autophagy within and between these pathways based on pathway crosstalk analysis. The method of pathway analysis based on global influence was applied to find the feature mRNAs involved in the crosstalk between autophagy and other AD-related pathways. Subsequently, the weighted gene coexpression network analysis was used to construct a coexpression module of feature mRNAs and differential long non-coding RNAs. Finally, based on two autophagy-related crosstalk genes (CD40 and SMAD7), we constructed a prognosis model by multivariate Cox regression, which could predict the overall survival of AD patients with medium-to-high accuracy. In conclusion, we provided an effective method for extracting autophagy-related significant genes based on pathway crosstalk in AD. We found the biomarkers valuable to the AD prognosis, which may also play an essential role in the development and treatment of AD.

LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy

LC3/ATG8 has long been appreciated to play a central role in autophagy, by which a variety of cytoplasmic materials are delivered to lysosomes and eventually degraded. However, information on the molecular functions of LC3 in RNA biology is very limited. Here, we show that LC3B is an RNA-binding protein that directly binds to mRNAs with a preference for a consensus AAUAAA motif corresponding to a polyadenylation sequence. Autophagic activation promotes an association between LC3B and target mRNAs and triggers rapid degradation of target mRNAs in a CCR4-NOT–dependent manner before autolysosome formation. Furthermore, our transcriptome-wide analysis reveals that PRMT1 mRNA, which encodes a negative regulator of autophagy, is one of the major substrates. Rapid degradation of PRMT1 mRNA by LC3B facilitates autophagy. Collectively, we demonstrate that LC3B acts as an RNA-binding protein and an mRNA decay factor necessary for efficient autophagy.

Role of BAG3 in bovine Deltapapillomavirus-mediated autophagy.

Bovine Deltapapillomavirus genus (δPV), comprises four members that are highly pathogenic and are frequently associated with bladder tumors of adult cattle and water buffaloes. In particular, bovine δPV-2 and δPV-13 are commonly found in urinary bladder tumors in adult large ruminants reared fully or partially on hilly/mountain pasturelands rich in bracken fern (Pteridium spp.) as the urinary bladder of the herbivores is the specific target for bracken genotoxins such as ptaquiloside (PT). PT is a sesquiterpenoid responsible for alkylation of adenine of codon 61 of gene H-Ras, which results in Glutamine 61 substitution that is essential for guanosine triphosphate (GTP) hydrolysis. Glutamine substitution at position 61 impairs the intrinsic GTPase activity. Therefore, active GTP-bound conformations (Ras-GTP) accumulate in cells, thereby causing abnormal cell proliferation and differentiation. The aim of the present study is to stress how bovine δPVs upregulate different forms of selective autophagy, of which BAG3 is a key player. BAG3 plays a central role in autophagy and acts as a multifunctional hub for an interaction network at the cytosolic and mitochondrial level. BAG3 is a functional partner of bovine δPV E5 oncoprotein and forms a complex with molecular chaperones Hsc70/Hsp70/Hsp8B and with cochaperone CHIP. BAG3 interacts with Synpo2. It is believed that this interaction has a crucial role for autophagosome (mitophagosome) formation. Furthermore, in urothelial cells infected by bovine δPVs, BAG3 interacts with parkin and some receptors such as BNIP3/FUNDC1, which suggests that BAG3 is involved in both parkin-dependent and -independent mitophagy that appear upregulate in bladder carcinogenesis of cattle induced by bovine δPVs. Surprisingly, BAG3 interacts also with ERAS, a protein encoded by the ERas gene, a novel member of the RAS family. Unlike in humans, the ERas gene is a functional gene in the cells of adult cattle, and it appears to play a role in bovine BAG3-mediated selective autophagy, including mitophagy observed in urothelial cells spontaneously infected with bovine papillomavirus.

Adding Some "Splice" to Stress Eating: Autophagy, ESCRT and Alternative Splicing Orchestrate the Cellular Stress Response.

Autophagy is a widely studied self-renewal pathway that is essential for degrading damaged cellular organelles or recycling biomolecules to maintain cellular homeostasis, particularly under cellular stress. This pathway initiates with formation of an autophagosome, which is a double-membrane structure that envelopes cytosolic components and fuses with a lysosome to facilitate degradation of the contents. The endosomal sorting complexes required for transport (ESCRT) proteins play an integral role in controlling autophagosome fusion events and disruption to this machinery leads to autophagosome accumulation. Given the central role of autophagy in maintaining cellular health, it is unsurprising that dysfunction of this process is associated with many human maladies including cancer and neurodegenerative diseases. The cell can also rapidly respond to cellular stress through alternative pre-mRNA splicing that enables adaptive changes to the cell’s proteome in response to stress. Thus, alternative pre-mRNA splicing of genes that are involved in autophagy adds another layer of complexity to the cell’s stress response. Consequently, the dysregulation of alternative splicing of genes associated with autophagy and ESCRT may also precipitate disease states by either reducing the ability of the cell to respond to stress or triggering a maladaptive response that is pathogenic. In this review, we summarize the diverse roles of the ESCRT machinery and alternative splicing in regulating autophagy and how their dysfunction can have implications for human disease.

Molecular characterization and role in virus infection of Beclin-1 in large yellow croaker (Larimichthys crocea)

Beclin-1, the ortholog of yeast autophagy-related gene 6 (Atg6), has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, and lifespan extension. However, understanding of function of fish Beclin-1 is limited now. In this study, the complete Beclin-1 cDNA of large yellow croaker Larimichthys crocea (LcBeclin-1) was cloned, whose open reading frame (ORF) is 1344 bp long and encodes a protein of 447 amino acids (aa). The deduced LcBeclin-1 possesses a typical Bcl-2 homology domain 3(BH3) and an APG6 domain that contains a central coiled-coil domain (CCD, residues 174 to 231) and a C-terminal evolutionarily conserved domain (ECD, residues 241 to 334). LcBeclin-1 shared a high amino acid identity of 81.66–98.66% with reported Beclin-1 molecules from other vertebrate species. LcBeclin-1 gene was constitutively expressed in all tissues tested, with the highest levels in heart. LcBeclin-1 transcripts were also detected in primary head kidney granulocytes (PKGs), primary head kidney macrophages (PKMs), primary head kidney leukocytes (PKLs), and large yellow croaker head kidney cell line (LYCK), and were significantly upregulated by poly (I:C) in PKMs and LYCK cells. Subcellular localization showed that LcBeclin-1 was evenly distributed in the cytoplasm and nucleus of LYCK cells. Overexpression of LcBeclin-1 significantly increased the replication of SVCV, as evidenced by increased severity of the cytopathic effects, enhanced viral titre, and upregulated transcriptional levels of viral genes. Further studies showed that LcBeclin-1 induced the occurrence of autophagy in LYCK cells. Additionally, LcBeclin-1 also decreased the expression levels of large yellow croaker interferons (IFNs; IFNc, IFNd, and IFNh), interferon regulatory factor 3 (IRF3) and IRF7, IFN-stimulated genes (ISGs; Mx, PKR, and Viperin) in LYCK cells. All these data suggest that LcBeclin-1 promoted the viral replication possibly by inducing autophagy or negatively modulating IFN response, which will help us to further understand the function of fish Beclin-1.

Molluscan Beclin-1 is involved in the innate immune response by regulating the autophagosomes formation in Crassostrea hongkongensis

Beclin-1 plays a central role in autophagy, which is well documented in mammals. However, the identity and function of Beclin-1 remain largely unclear in mollusks. In this study, a Beclin-1 gene (ChBeclin-1) was identified and characterized from Crassostrea hongkongensis. The full length of ChBeclin-1 cDNA encoded a predicted 442-amino acid protein with a deduced molecular weight of 51.11 kDa and a theoretical isoelectric point of 4.71. The putative amino acid sequence of ChBeclin-1 contained a typical Bcl-2 homology domain 3 (BH3), a coiled-coil domain (CCD), and an Atg6 domain. Phylogenetic analysis showed that ChBeclin-1 was clustered with Beclin-1 from other mollusks such as Crassostrea gigas, Crassostrea virginica, and Mizuhopecten yessoensis. ChBeclin-1 mRNA was constitutively expressed in all detected tissues, with the highest relative expression in the gills. Additionally, significant inductions in the mRNA level of ChBeclin-1 were observed after bacterial stimulations, indicating its involvement in the innate immune response. The knockdown of ChBeclin-1 via RNA interference impeded the formation of autophagosomes, denoting ChBeclin-1 plays a role in autophagy. Moreover, in subcellular localization and dual-luciferase reporter assays, ChBeclin-1 protein was found to be localized mainly in the endoplasmic reticulum, and its overexpression suppressed the transcriptional activity of an NF-κB reporter gene in a dose-dependent manner in HEK 293T cells. Collectively, these findings provide experimental evidence of a functional Beclin-1 in autophagy and demonstrate its involvement in the innate immunity of C. hongkongensis.

A Proteomic Atlas of Cardiac Amyloid Plaques

BackgroundIn vivo mechanisms of amyloid clearance and cardiac tissue damage in cardiac amyloidosis are not well understood. ObjectivesWe aimed to define and quantify the amyloid plaque proteome in cardiac transthyretin amyloidosis (ATTR) and light chain amyloidosis (AL) and identify associations with patient characteristics and outcomes. MethodsA proteomics approach was used to identify all proteins in cardiac amyloid plaques, and to compare both normal and diseased controls. All proteins identified within amyloid plaques were defined as the expanded proteome; only proteins that were enriched in comparison to normal and disease controls were defined as the amyloid-specific proteome. ResultsProteomic data from 292 patients with ATTR and 139 patients with AL cardiac amyloidosis were included; 160 and 161 unique proteins were identified in the expanded proteomes, respectively. In the amyloid-specific proteomes, we identified 28 proteins in ATTR, 19 in AL amyloidosis, with 13 proteins overlapping between ATTR and AL. ATTR was characterized by a higher abundance of complement and contractile proteins and AL by a higher abundance of keratins. We found that the proteome of kappa AL had higher levels of clusterin, a protective chaperone, and lower levels of light chains than lambda despite higher levels of circulating light chains. Hierarchical clustering identified a group of patients with worse survival in ATTR, characterized by high levels of PIK3C3, a protein with a central role in autophagy. ConclusionsCardiac AL and ATTR have both common and distinct pathogenetic mechanisms of tissue damage. Our findings suggest that autophagy represents a pathway that may be impaired in ATTR and should be further studied.

A multitude of signaling pathways associated with Alzheimer's disease and their roles in AD pathogenesis and therapy.

The exact molecular mechanisms associated with Alzheimer's disease (AD) pathology continue to represent a mystery. In the past decades, comprehensive data were generated on the involvement of different signaling pathways in the AD pathogenesis. However, the utilization of signaling pathways as potential targets for the development of drugs against AD is rather limited due to the immense complexity of the brain and intricate molecular links between these pathways. Therefore, finding a correlation and cross-talk between these signaling pathways and establishing different therapeutic targets within and between those pathways are needed for better understanding of the biological events responsible for the AD-related neurodegeneration. For example, autophagy is a conservative cellular process that shows link with many other AD-related pathways and is crucial for maintenance of the correct cellular balance by degrading AD-associated pathogenic proteins. Considering the central role of autophagy in AD and its interplay with many other pathways, the finest therapeutic strategy to fight against AD is the use of autophagy as a target. As an essential step in this direction, this comprehensive review represents recent findings on the individual AD-related signaling pathways, describes key features of these pathways and their cross-talk with autophagy, represents current drug development, and introduces some of the multitarget beneficial approaches and strategies for the therapeutic intervention of AD.

Multiple Functions of ATG8 Family Proteins in Plant Autophagy.

Autophagy is a major degradation process of cytoplasmic components in eukaryotes, and executes both bulk and selective degradation of targeted cargos. A set of autophagy-related (ATG) proteins participate in various stages of the autophagic process. Among ATGs, ubiquitin-like protein ATG8 plays a central role in autophagy. The ATG8 protein is conjugated to the membrane lipid phosphatidylethanolamine in a ubiquitin-like conjugation reaction that is essential for autophagosome formation. In addition, ATG8 interacts with various adaptor/receptor proteins to recruit specific cargos for degradation by selective autophagy. The ATG8-interacting proteins usually contain the ATG8-interacting motif (AIM) or the ubiquitin-interacting motif (UIM) for ATG8 binding. Unlike a single ATG8 gene in yeast, multiple ATG8 orthologs have been identified in the plant kingdom. The large diversity within the ATG8 family may explain the various functions of selective autophagy in plants. Here, we discuss and summarize the current view of the structure and function of ATG8 proteins in plants.

Heterozygous disruption of beclin 1 mitigates arsenite-induced neurobehavioral deficits via reshaping gut microbiota-brain axis

Gut microbiota is intimately involved in numerous aspects of human health. Arsenite expouse can perturb gut microbiota and is linked to increased susceptibility of individual to arsenite-related diseases. However, how microbiome factors influence arsenite-induced neurotoxicity remains largely unknown. In this study, after treating of healthy adult female mice with arsenite via drinking water for 6 months, our results clearly revealed that chronic arsenite exposure not only perturbed the composition of gut microbiota but also caused neurobehavioral dysfunctions, which manifested by learning and memory deficits and anxiety-like behavior. Given that the overactive autophagy directly leads to gut pathological changes, we further assessed whether inhibiton of autophagy by genetic mean could reverse arsenite-induced neurobehavioral dysfunctions. Our results illustrated for the first time that heterozygous disruption of beclin 1, which played a central role in autophagy, alleviated the perturbation of gut microbiome phenotypes induced by arsenite, and ultimately leading to the improvement of neurobehavioral deficits through gut-brain communication. These findings provide a new clue that regulation of autophagy is a potential approach for probing the functional impacts of arsenite on the gut microbiome, and it also may be severed as a way for protection strategies against arsenite neurotoxicity.

Deacetylation of Nuclear LC3 Drives Autophagy Initiation under Starvation

Shuttling of macromolecules between different cellular compartments helps regulate the timing and extent of different cellular activities. Here, we report that LC3, a key initiator of autophagy that cycles between the nucleus and cytoplasm, becomes selectively activated in the nucleus during starvation through deacetylation by the nuclear deacetylase Sirt1. Deacetylation of LC3 at K49 and K51 by Sirt1 allows LC3 to interact with the nuclear protein DOR and return to the cytoplasm with DOR, where it is able to bind Atg7 and other autophagy factors and undergo phosphatidylethanolamine conjugation to preautophagic membranes. The association of deacetylated LC3 with autophagic factors shifts LC3's distribution from the nucleus toward the cytoplasm. Thus, an acetylation-deacetylation cycle ensures that LC3 effectively redistributes in an activated form from nucleus to cytoplasm, where it plays a central role in autophagy to enable the cell to cope with the lack of external nutrients.

New TB treatments hiding in plain sight.

As tuberculosis (TB) toll is revised upward according to the WHO's last estimates, the lack of vaccine strategy and the lengthy antibiotic treatments that unfortunately promote the emergence of drug resistance are a major set back in the fight against this pathogen. In this issue of EMBO Molecular Medicine, Schiebler et al (Mtb) propose a novel and compelling new approach to target Mycobacterium tuberculosis (Mtb) by pharmacologically stimulating intracellular mycobacteria clearance through autophagy.

Molecular characterization and expression analysis of the autophagic gene Beclin 1 from the purse red common carp (Cyprinus carpio) exposed to cadmium

Beclin 1, the mammalian orthologue of yeast Atg6, has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, lifespan extension, etc. However, the relevant study about Beclin 1 is rare in fish compared with mammals. In this study, we isolated Beclin 1 gene from the kidney tissue of common carp (Cyprinus carpio) using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of cloned Beclin 1 comprised 447 amino acids, which showed approximately 80.7% identity and 88.9% similarity to human Beclin 1. It possessed a typical Bcl-2 homology domain 3 (BH3) and an evolutionarily conserved domain (ECD). Phylogenetic analysis demonstrated that common carp Beclin 1 formed a clade with zebrafish Beclin 1. To explore the relationship between Beclin 1 and cadmium (Cd)-induced injury, a Cd exposure experiment was conducted. The result showed that Cd content was significantly increased in a dose-dependent manner in kidney after Cd exposure. Swelling and vacuolation of renal tubular epithelial cells, and glomerular hyalinization were observed. Renal leukocyte infiltration was diffusely distributed in the interstitial tissue. Real-time quantitative RT-PCR analysis revealed that the mRNA transcript level of Beclin 1 was markedly up-regulated in a dose-dependent and time-dependent manner after exposure to Cd. Similarly, Western blot analysis indicated that its protein level was significantly elevated in a dose-dependent manner after Cd treatment. All the results indicate that the common carp Beclin 1 gene may play a regulatory role against Cd toxicity.

Inclusion body formation, macroautophagy, and the role of HDAC6 in neurodegeneration

The failure to clear misfolded or aggregated proteins from the cytoplasm of nerve cells and glia is a common pathogenic event in a variety of neurodegenerative disorders. This might be causally related to defects in the major proteolytic systems, i.e., the ubiquitin-proteasomal system and the autophagic pathway. Large protein aggregates and defective organelles are excluded from the proteasome. They can be degraded only by macroautophagy, which is a highly selective process. It requires p62 to act as a bridge connecting ubiquitinated protein aggregates and autophagosomes, and the tubulin deacetylase histone deacetylase 6 (HDAC6). HDAC6 has recently been identified as a constituent in Lewy bodies of Parkinson disease and glial cytoplasmic inclusions of multiple system atrophy. It is considered a sensor of proteasomal inhibition and a cellular stress surveillance factor, and plays a central role in autophagy by controlling the fusion process of autophagosomes with lysosomes. Upon proteasomal inhibition, HDAC6 is relocated and recruited to polyubiquitin-positive aggresomes. Tubulin acetylation is a major consequence of HDAC6 inhibition, and HDAC6 inhibition restores microtubule (MT)-dependent transport mechanisms in neurons. This suggests the involvement of HDAC6 in neurodegenerative diseases. Furthermore, the protein tau seems to be a substrate for HDAC6. Tau acetylation impairs MT assembly and promotes tau fibrillization in vitro. It has been suggested that acetylation and phosphorylation of tau at multiples sites may act synergistically in the pathogenesis of tau fibrillization. In this review, we will survey the process of aggresome formation, macroautophagy and the role of autophagosomal proteins and HDAC6 in inclusion body formation.

Reconstitution of the Class III Phosphatidylinositol‐3‐kinase in autophagy

The class III phosphatidylinositol‐3‐kinase (PI3KC3) plays a central role in autophagy and endocytosis. How its function is differentially regulated in these processes remain elusive. We are the first group to purify PI3KC3 holocomplex in human cells, in which we found seven subunits including Vps34, p150, Beclin 1, Barkor/Atg14(L), NRBF2, Rubicon and UVRAG. These proteins form two mutually exclusive subcomplexes sharing Vps34, p150 and Beclin 1. Barkor and NRBF2 regulate PI3KC3 activity on autophagosome membrane, while UVRAG and Rubicon control PI3KC3 activity on endosome membrane. We have reconstituted the biochemical activity of autophagic PI3KC3 activity in vitro, and explored their functions in membrane curvature sensing and membrane tethering/hemifusion.

Su1163 The Loss of Beclin-1 Expression Correlates With Progression to Esophageal Adenocarcinoma and Autophagy Resistance: the Role of Bile Acids

Background and Aims: Autophagy is a normal physiological mechanism for the degradation of cellular proteins and organelles. Excessive autophagy induced by cellular stress leads to cell death. However, the exact role of autophagy in cancer is not completely clear. Beclin1 plays a central role in the regulation of autophagy. The major goal of our study was to evaluate the role of Beclin-1 and autophagy and the effect of bile acids in the esophageal carcinogenesis. Barrett's esophagus (BE) is a premalignant lesion of distal esophagus associated with an increased risk for the development of esophageal adenocarcinoma (EAC). Methods: Beclin-1 expression was evaluated using immunohistochemistry, immunoblotting, or RTPCR in (1) biopsies obtained from 62 patients with BE or EAC, (2) tissues from a rat model of BE and EAC, and (3) esophageal cell lines. Since reflux of bile acids is important in esophageal carcinogenesis, the effect of acute and chronic exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression was evaluated. Autophagy was assessed by electron microscopy or by transfection with GFP-LC3 plasmid and calculating the percentage of GFP-LC3 positive cells with punctuate pattern. Results: Our studies using human biopsies and rat tissues showed that Beclin-1 expression was high in squamous epithelium, while its expression was low in BE and EAC. A similar pattern was observed in esophageal cell lines. HET-1A cells (derived from normal squamous epithelium) showed high levels of Beclin-1, but lower levels of Beclin-1 were found in BE cells (CP-A, CP-C) and EAC cells (OE33 cells). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy in CP-A cells. In contrast, chronic exposure to DCA did not result in any alteration of Beclin-1 or autophagy. To demonstrate that Beclin-1 plays central role in autophagy we used siRNA to decrease Beclin-1 expression and then autophagy was evaluated after treatment with DCA. No increase in autophagy was detected compared to untreated control CP-A cells. Conclusions: In summary, our data suggests that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance. We propose that autophagy resistance in combination with apoptosis resistance induced by chronic exposure to bile acids contributes to EAC development.

Su1164 Changes in Esophageal Microbiome During the Development of Barrett's Esophagus in a Rat Model

Background and Aims: Autophagy is a normal physiological mechanism for the degradation of cellular proteins and organelles. Excessive autophagy induced by cellular stress leads to cell death. However, the exact role of autophagy in cancer is not completely clear. Beclin1 plays a central role in the regulation of autophagy. The major goal of our study was to evaluate the role of Beclin-1 and autophagy and the effect of bile acids in the esophageal carcinogenesis. Barrett's esophagus (BE) is a premalignant lesion of distal esophagus associated with an increased risk for the development of esophageal adenocarcinoma (EAC). Methods: Beclin-1 expression was evaluated using immunohistochemistry, immunoblotting, or RTPCR in (1) biopsies obtained from 62 patients with BE or EAC, (2) tissues from a rat model of BE and EAC, and (3) esophageal cell lines. Since reflux of bile acids is important in esophageal carcinogenesis, the effect of acute and chronic exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression was evaluated. Autophagy was assessed by electron microscopy or by transfection with GFP-LC3 plasmid and calculating the percentage of GFP-LC3 positive cells with punctuate pattern. Results: Our studies using human biopsies and rat tissues showed that Beclin-1 expression was high in squamous epithelium, while its expression was low in BE and EAC. A similar pattern was observed in esophageal cell lines. HET-1A cells (derived from normal squamous epithelium) showed high levels of Beclin-1, but lower levels of Beclin-1 were found in BE cells (CP-A, CP-C) and EAC cells (OE33 cells). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy in CP-A cells. In contrast, chronic exposure to DCA did not result in any alteration of Beclin-1 or autophagy. To demonstrate that Beclin-1 plays central role in autophagy we used siRNA to decrease Beclin-1 expression and then autophagy was evaluated after treatment with DCA. No increase in autophagy was detected compared to untreated control CP-A cells. Conclusions: In summary, our data suggests that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance. We propose that autophagy resistance in combination with apoptosis resistance induced by chronic exposure to bile acids contributes to EAC development.

Su1166 Pax9 is Critical for Esophageal Epithelial Development

Background and Aims: Autophagy is a normal physiological mechanism for the degradation of cellular proteins and organelles. Excessive autophagy induced by cellular stress leads to cell death. However, the exact role of autophagy in cancer is not completely clear. Beclin1 plays a central role in the regulation of autophagy. The major goal of our study was to evaluate the role of Beclin-1 and autophagy and the effect of bile acids in the esophageal carcinogenesis. Barrett's esophagus (BE) is a premalignant lesion of distal esophagus associated with an increased risk for the development of esophageal adenocarcinoma (EAC). Methods: Beclin-1 expression was evaluated using immunohistochemistry, immunoblotting, or RTPCR in (1) biopsies obtained from 62 patients with BE or EAC, (2) tissues from a rat model of BE and EAC, and (3) esophageal cell lines. Since reflux of bile acids is important in esophageal carcinogenesis, the effect of acute and chronic exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression was evaluated. Autophagy was assessed by electron microscopy or by transfection with GFP-LC3 plasmid and calculating the percentage of GFP-LC3 positive cells with punctuate pattern. Results: Our studies using human biopsies and rat tissues showed that Beclin-1 expression was high in squamous epithelium, while its expression was low in BE and EAC. A similar pattern was observed in esophageal cell lines. HET-1A cells (derived from normal squamous epithelium) showed high levels of Beclin-1, but lower levels of Beclin-1 were found in BE cells (CP-A, CP-C) and EAC cells (OE33 cells). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy in CP-A cells. In contrast, chronic exposure to DCA did not result in any alteration of Beclin-1 or autophagy. To demonstrate that Beclin-1 plays central role in autophagy we used siRNA to decrease Beclin-1 expression and then autophagy was evaluated after treatment with DCA. No increase in autophagy was detected compared to untreated control CP-A cells. Conclusions: In summary, our data suggests that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance. We propose that autophagy resistance in combination with apoptosis resistance induced by chronic exposure to bile acids contributes to EAC development.

Abstract C150: CaMKKβ-mediated phosphorylation of Beclin 1 regulates autophagy and cell differentiation in neuroblastoma cells.

Abstract Autophagy is an evolutionarily conserved degradative pathway by which components of the cells are degraded in the lysosome in response to stress. Beclin 1 plays a central role in autophagy. Pioneering work has indicated that the increase in cytosolic Ca(2+) can induce autophagy via the CaMKKβ-mediated activation of AMPK. Here, we demonstrate a novel signaling pathway in which CaMKKβ regulates autophagy and differentiation. We found that Ca(2+) mobilizing agents (ionomycin and EB1089) induced phosphorylation of Beclin 1 and autophagy through CaMKKβ-dependent pathway. Using In vitro kinase assay we demonstrated that CaMKKβ directly phosphorylated Beclin 1 Ser90. Moreover, autophagy induced by ionomycin or EB1089 contributed to degradation of Id1 (inhibitor of differentiation). The inhibitors of autophagy including Bafilomycin A1 and chloroquine and RNA interference of autophagy-associated gene atg 5 markedly increased the protein levels of Id1 and abrogated neuroblastoma cell differentiation. Taken together, CaMKKβ promoted autophagy by directly phosphorylating Beclin 1 at Ser90, and thus induced cell differentiation in neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C150.